Compositions and methods for immunization against drug resistant acinetobacter baumannii

ABSTRACT

The present invention provides vaccine compositions comprising OmpA, or antigenic fragments thereof, and related methods of active immunization against  A. baumannii  infection. The invention also provides antibodies and antigen-binding parts thereof that specifically bind to OmpA, and related methods of passive immunization against  A. baumannii  infection. The compositions and methods of the invention are useful for preventing or treating  A. baumannii  infections, including those caused by strains resistant to carbapenems and all other antibiotics except colistin or tigecycline, also referred to as extreme drug resistant (XDR)  A. baumannii  infections, and those resistant to every FDA approved antibiotic, also referred to as pan-drug resistant (PDR)  A. baumannii  infections.

This application is a continuation of United States Non-provisionalapplication Ser. No. 13/470,177, filed May 11, 2012, which claims thebenefit of priority of U.S. Provisional application Ser. No. 61/486,177,filed May 13, 2011, the entire contents of each of which areincorporated herein by reference.

This invention was made with government support under grant number PHSR01 AI081719, AI077681, and AI072052 awarded by NIH/NIAID. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Antibiotic resistance is recognized as one of the greatest threats tohuman health on the planet (2009; Choffnes et al., AntibioticResistance: Implications for Global Health and Novel InterventionStrategies, The National Academic Press, Washington, D.C., (2010);Smolinski et al., Microbial Threats to Health: Emergence, Detection, andResponse, The Institute of Medicine, Washington D.C., (2003); Spellberget al., Clin Infect Dis 52(55):397-428 (2011); Spellberg et al., ClinInfect Dis 46:155-164 (2008); Walker et al., Science 325-1345-1346(2009). In the last decade, Acinetobacter baumannii has emerged as oneof the most common and highly antibiotic-resistant pathogens in theUnited States (US) and throughout the world (Doi et al., Emerg InfectDis 15:980-982 (2009); Higgins et al., J Antimicrob Chemother 65-233-238(2010); Perez et al., Antimicrob Agents Chemother 51:3471-3484 (2007).Indeed, 50-70% of A. baumannii clinical isolates are now extensivelydrug resistant (XDR; i.e. resistant to carbapenems and all otherantibiotics except colistin or tigecycline), reflecting a >15-foldincrease in just the past 10 years (Dizbay et al., Scand J Infect Dis(2010); Hidron et al., Infect Control Hosp Epidemiol 29:996-1011 (2008);Hoffmann et al., Infect Control Hosp Epidemiol 31:196-197 (2010); Kallenet al., Infect Control Hosp Epidemiol 31:528-531 (2010); Lautenbach etal., Infect Control Hosp Epidemiol 30:1186-1192 (2009); Mera et al.,Drug Resist 16:209-215 (2010); Perez et al., Am J Infect Control38:63-65 (2010); Rosenthal et al., Am J Infect Control 38:95-104 e102(2010). Infections caused by carbapenem-resistant, XDR A. baumannii areassociated with prolonged hospitalization, tremendous health care costs,and high rates of death despite treatment (Doi et al., Emerg Infect Dis15:980-982 (2009); Falagas et al., Int J Antimicrob Agents 32:450-454(2008); Gordon and Wareham, J Antimicrob Chemother 63:775-780 (2009);Lautenbach et al., Infect Control Hosp Epidemiol 30:1186-1192 (2009);Metan et al., Eur J Intern Med 20:540-544 (2009); Park et al., DiagnMicrobiol Infect Dis 64:43-51 (2009); Perez et al., Am J Infect Control38:63-65 (2007); Sunenshine et al., Emerg Infect Dis 13:97-103 (2007).Indeed, bloodstream infections caused by XDR A. baumannii cause >50-60%mortality rates despite antibiotic therapy (Gordon and Wareham, JAntimicrob Chemother 63:775-780 (2009); Metan et al., Eur J Intern Med20:540-544 (2009); Munoz-Price et al., Infect Control Hosp Epidemiol1(10):1057-62 (2010); Park et al., Diagn Microbiol Infect Dis 64:43-51(2009); Tseng et al., Diagn Microbiol Infect Dis 59:181-190 (2007). Amajor reason for these high mortality rates is that XDR A. baumanniiinfections are treatable only with suboptimal second-line antibacterialagents, such as tigecycline and colistin. Even more concerning is theincreasing resistance of A. baumannii to both colistin and tigecycline(Adams et al., Antimicrob Agents Chemother 53:3628-3634 (2009); Doi etal., Emerg Infect Dis 15:980-982 (2009); Falagas et al., Int JAntimicrob Agents 32:450-454 (2008); Hernan et al., Diagn MicrobiolInfect Dis 65:188-191 (2009); Livermore et al., Int J Antimicrob Agents35:19-24 (2010); Park et al., Diagn Microbiol Infect Dis 64:43-51(2009); Valencia et al., Infect Control Hosp Epidemiol 30:257-263(2009); Wang and Dowzicky, Diagn Microbiol Infect Dis 68:73-79 (2010).Such pan-drug resistant (PDR) A. baumannii infections are resistant toevery FDA approved antibiotic, and are hence untreatable.

New methods to prevent such XDR/PDR A. baumannii infections arecritically needed, especially since no new drugs to treat theseinfections are in the antibacterial pipeline for the coming decade(Boucher et al., Clin Infect Dis 48:1-12 (2009); Spellberg et al., ClinInfect Dis 46:155-164 (2008). Since risk factors for A. baumanniiinfections are understood (Beavers et al., 2009; Caricato et al.,Intensive Care Med 35:1964-1969 (2009); D'Agata et al., Infect ControlHosp Epidemiol 21:588-591 (2000); Furniss et al., J Burn Care Rehabil26:405-408 (2005); Metan et al., Eur J Intern Med 20:540-544 (2009);Zakuan et al., Trop Biomed 26:123-129 (2009), vaccination of acutelyat-risk patients is a promising method to prevent such infections, andantibody-based immunotherapy has promise to improve outcomes frominfection.

SUMMARY OF INVENTION

The present invention provides vaccine compositions comprising OmpA, orantigenic fragments thereof, and related methods of active immunizationagainst A. baumannii infection. The invention also provides antibodiesand antigen-binding fragments thereof that specifically bind to OmpA,and related methods of passive immunization against A. baumanniiinfection. The compositions and methods of the invention are useful forpreventing or treating A. baumannii infections, including those causedby strains resistant to carbapenems and all other antibiotics exceptcolistin or tigecycline, also referred to as extreme drug resistant(XDR) A. baumannii infections, and those resistant to every FDA approvedantibiotic, also referred to as pan-drug resistant (PDR) A. baumanniiinfections.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. A. baumannii infection induces specific humoral immune response.Ten mice were infected with ATCC 17978 (top) and 2 mice each wereinfected with clinical isolates from Harbor-UCLA Medical Center (HUMC)(bottom). Paired pre-immune & immune serum IgG anti-A. baumannii cellmembrane protein titers are shown.

FIG. 2. 2 Dimensional PAGE-IEF gels and western blots of cell membraneprotein extracts of A. baumannii clinical isolates. (A) Membrane proteinpreparations from A. baumanni clinical strains (ATCC 17978 & HUMC1, 4,5, 6, & 12) were run on 2 D gels stained with Coomassie Blue. (B)Western blots of those 2D gels were stained with paired sera obtainedfrom mice before infection (pre-serum) and after recovery fromnon-lethal iv infection (post-serum) with A. baumannii. Spots uniquelyidentified by post-immune serum were seen at conserved locations. Spotsselected for protein identification by MALDI-TOF analysis are markedwith white arrows.

FIG. 3. A. baumannii infection induces specific anti-rOmpA antibodyresponse. Ten mice were infected with ATCC 17978 (top) and 2 mice eachwere infected with clinical isolates from Harbor-UCLA Medical Center(HUMC) (bottom). Paired pre-immune & immune serum IgG anti-rOmpA cellmembrane protein titers are shown.

FIG. 4. OmpA sequence alignment across clinical isolates used in thecurrent study. OmpA is >99% homologous at the amino acid level acrosssix clinical isolates of A. baumannii harvested 58 years (1951-2009)apart (SEQ ID NOS:1-6), including carbapenem-susceptible andcarbapenem-resistant strains.

FIG. 5. Vaccination with rOmpA protected mice from lethal A. baumanniiinfection in a disseminated sepsis model. A) Survival of retired breeder(>6 mo) diabetic Balb/c mice vaccinated with 100 μg of rOmpA or aluminumhydroxide (AlOH₃) adjuvant alone (n=6 adjuvant control and 8 vaccinated)and infected with 2×10⁷ A. baumannii HUMC1. B) Survival of juvenile(8-10 weeks) diabetic Balb/c mice vaccinated with 3, 30, or 100 μg ofrOmpA or adjuvant alone (n=10 adjuvant, 12 mice in the 3 μg group, 13mice in the 30 μg group, and 10 mice in the 100 μg group) and infectedwith 2×10⁷ A. baumannii HUMC1. C) Tissue bacterial burden in diabeticmice (n=10 control and 13 vaccinated) infected with 10⁷ A. baumanniiHUMC1. *p<0.05 vs. adjuvant control; **p<0.05 vs. adjuvant control andvs. 3 μg group.

FIG. 6. Anti-rOmpA antibody titers correlated with survival in infectedmice. A) Survival of juvenile diabetic Balb/c mice vaccinated with 3 μgof rOmpA or adjuvant alone (n=20 mice per group from 2 experiments) andinfected with 1.4 or 1.6×10⁷ A. baumannii HUMC1 in the sequentialexperiments. The experiments were terminated at 28 days with allremaining mice appearing clinically well. B) Antibody titers ofindividual vaccinated and control mice vs. day of death).

FIG. 7. Passive immunization with rOmpA immune serum protected recipientmice from lethal infection. Survival of mice (n=10 per group) treated ipwith immune (from OmpA vaccinated) or adjuvant control serum beforetail-vein infection with A. baumannii HUMC1. *p=<0.0001 vs. non-immuneserum. B) Opsonophagocytic killing of A. baumannii HUMC1 duringincubation of macrophages with immune (from OmpA vaccinated mice) ornon-immune (from adjuvant treated mice) serum. *p<***vs. control.

FIG. 8. Antibody titers induced by various doses of rOmpA or adjuvantalone. A) Balb/c mice (n=11 per group from 3 separate experiments) werevaccinated with one of 3 doses of vaccine or adjuvant alone. IgG titersfrom individual mice and the median titers (horizontal bars) for eachgroup are shown. B) IgM and IgG subtype titers measured by ELISA fromvaccinated or control mice. *p<0.05 vs. adjuvant alone; **p<0.05 vs.adjuvant alone and vs. 3 μg dose.

FIG. 9. Splenocyte cytokine production stimulated by rOmpA. A) IFN-γ,IL-4, or IL-17A production by splenocytes from vaccinated or controlmice (n=8 per group from 2 experiments) stimulated for 48 h with rOmpAmeasured by ELISpot. B) Ratio of IFN-γ:IL-4 produced by splenocytes fromindividual mice. Median and interquartile ranges are shown. *p<0.05 vs.adjuvant control. **p<0.05 vs. 3 and 30 μg dose, and vs. adjuvantcontrol.

FIG. 10. T cell epitopes stimulate distinct cytokine profiles.Splenocytes were harvested from vaccinated Balb/c mice and stimulatedwith 5 μg/ml of individual, overlapping 15mer peptides for 48 hours inELISpot plates. Graphed are the means of 2 mice per group each run induplicate. The lower bound of the Y axis is set at the third quartile ofresponses across all peptides.

FIG. 11. Peptide epitope mapping of OmpA using polyclonal immune serumfrom OmpA-vaccinated mice. Each spot contains a peptide recognized byimmune serum. The immunogenic epitopes shown are: 1. SPOTs 86-92, aminoacids 265PRKLNERLSLARANSV280 (SEQ ID NO:7); 2. SPOTs 102-105, aminoacids 307ADNKTKEGRAMNR319 (SEQ ID NO:8); 3. SPOTs 107-108, amino acids319RRVFATITGSRTV331 (SEQ ID NO:9); 4. SPOTs 40-41, amino acids121KYDFDGVNRGTRG133 (SEQ ID NO:10).

FIG. 12. In silica model of OmpA protein. The model was built using theSwiss-Model automated protein structure homology-modeling serveraccessible via the ExPASy web server, or from the program DeepView(Swiss Pdb-Viewer). Major immunogenic epitopes are color-coded (seeadjacent text).

FIG. 13. Comparison of sequences of known B and T cell epitopes to A.baumannii OmpA sequences from ATCC17978 and HUMC strains used to infectmice. CLUSTAL format alignment by MAFFT (v6.821b) (SEQ ID NOS:1-6 and11, respectively). Yellow highlight=T cell epitopes (amino acids 1-18,51-65, 151-153 and 221-235), Blue=B cell epitopes (amino acids 26-32,91-130, 166, 265-280 and 307-331), Green=T and B cell epitopes (aminoacids 19-25 and 154-165), Gray=mutation present in the prior art in themidst of B or T cell epitopes (amino acids 35F, 39N, 48M, 56T, 83I, 85V,119A, 124A, 128V, 129F, 131G, 137V, 141M, 151E, 153E, 156P, 1791, 184A,191G, 194H, 296A and 339N of SEQ ID NO:11).

FIG. 14. Immune serum from mice infected with A. baumannii generatesignificantly higher antibody titers to our patented OmpA sequence thanto protein made from a synthetic gene (SOmpA) based on the prior artsequence. Anti-OmpA ELISA was used to determine titers in immune serumdirected against protein made with our sequence (OmpA) vs. the prior artsequence (SOmpA). P value for the difference=0.002.

FIG. 15. Anti-OmpA MAb treats lethal A. baumannii infection. Mice (n=10per group from 2 experiments) were infected iv via the tail vein andtreated ip with 50 μg of MAb or isotype control antibody per mouse.*p<0.05 vs. control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of A.baumannii OmpA as an antigen target for an A. baumannii-targetedvaccine. The present invention provides vaccine compositions comprisingOmpA, or antigenic fragments thereof, and related methods of activeimmunization against A. baumannii infection. The invention also providesantibodies and antigen-binding parts thereof that specifically bind toOmpA, and related methods of passive immunization against A. baumanniiinfection. The compositions and methods of the invention are useful forpreventing or treating A. baumannii infections, including those causedby strains resistant to carbapenems and all other antibiotics exceptcolistin or tigecycline, also referred to as extreme drug resistant(XDR) A. baumannii infections, and those resistant to every FDA approvedantibiotic, also referred to as pan-drug resistant (PDR) A. baumanniiinfections.

As described herein, OmpA provides an antigen for an A.baumannii-targeted vaccine. As described in the examples, OmpA wasidentified as a vaccine based on humoral immunodominance duringinfection in mice. OmpA was highly conserved across multiple clinicalisolates, and shared minimal homology with the human proteome.

Over the past decade A. baumannii has emerged to become one of the mostantibiotic-resistant causes of infections all over the world, withunacceptably high resulting mortality rates. No new treatments capableof treating XDR/PDR A. baumannii are likely to become available duringthe coming decade and this invention provides novel strategies toprevent and treat such infections based on discovery of an antigen foran A. baumannii-targeted vaccine. rOmpA was identified as a vaccinebased on humoral immunodominance during infection in mice. OmpA washighly conserved across multiple clinical isolates, and shared minimalhomology with the human proteome. Substantial efficacy was seen inhighly and rapidly lethal murine models in immunocompromised, DKA micewhen administered with Al(OH)3 adjuvant, and will also be observed in arat model of aspiration pneumonia. Efficacy in two distinct models withAl(OH)3 demonstrates translatability of the vaccine candidate, sinceAl(OH)3 is one of the most widely used adjuvants in the world, and hasan established safety and efficacy record after dosing in millions ofpatients over more than a half century (Lindblad, Vaccine 22:3658-3668(2004); Lindblad, Immunol Cell Biol 82:497-505 (2004).

As exemplified herein, individual mouse antibody titers correlated withsurvival, and IgG titer cut-offs of ≧1:102,400 or 1:204,800 were highlyaccurate at predicting which mice survived. Furthermore, immune sera wasthe effector of vaccine-mediated protection, and was effective duringpassive immunization. It has been previously reported that A. baumanniiis resistant to complement-mediated killing (Kim et al., FEMS MicrobiolLett 301:224-231 (2009); King et al., FEMS Microbiol Lett 301:224-231(2009) which is concordant with the current study results.Immunization-induced protection against A. baumannii was mediated byenhancing opsonophagocytic killing of the organism. These results areconcordant with the fact that neutropenic mice are susceptible to A.baumannii infection (van Faassen et al., Infect Immun 75:5597-5608(2007) and the fact that superoxide-deficient, gp91phox−/− mice werehypersusceptible to A. baumannii intranasal infection (Qiu et al.,Infect Immun 75:5597-5608 (2009). Collectively, these results confirmthat enhanced uptake and killing of A. baumannii by antibody-basedopsonophagocytosis lead to more effective clearance of A. baumannii fromtissue.

A. baumannii OmpA has been found to have a variety of interestingbiological properties in model systems. For example, OmpA has been shownto bind to eukaryotic cells, translocate to the nucleus, and induce celldeath (Choi et al., Cell Microbiol 10:309-319 (2008); McConnell andPachon, Protein Expr Purif 77(1):98-103 (2010).

OmpA is a novel vaccine that can prevent XDR/PDR A. baumanniiinfections. As exemplified herein, efficacy has been established atfeasible doses with a translatable adjuvant.

The present invention provides a method of prophylactic or therapeutictreatment of A. baumannii infection in a mammalian subject, preferablyhuman, comprising administering to the subject an immunologicallyeffective amount of a A. baumannii OmpA vaccine composition, antibodycomposition or antiserum of the invention as described herein. In oneembodiment, the invention provides a method of prophylactic ortherapeutic treatment of A. baumannii infection in a subject, comprisingadministering to the subject an immunologically effective amount of avaccine composition comprising an A. baumannii outer membrane protein A(OmpA), or an antigenic fragment thereof. In a particular embodiment,the subject is a human.

The term “OmpA” or “A. baumannii OmpA” as used herein, means an outermembrane protein A of A. baumannii that corresponds to any of the aminoacid sequences shown in FIG. 4. The term also includes art-known OmpAamino acid sequences that are substantially similar in sequence,immunogenicity and function, including, for example, one or more of theOmpA sequences set forth in Table 1, which are incorporated herein byreference to their NCBI Accession.Version and gi sequence identifiers.An OmpA sequence of the invention can be, for example, at least 80percent, at least 85 percent, at least 87 percent, at least 88 percent,at least 89 percent, at least 90 percent, at least 91 percent, at least92 percent, at least 93 percent, at least 94 percent, at least 95percent, at least 96 percent, at least 97 percent, at least 98 percent,at least 99 percent, identical to a sequence set forth in FIG. 4. AnOmpA of the invention can, for example, be less than 360 amino acids inlength, less than 359 amino acids in length, less than 358 amino acidsin length, less than 357 amino acids in length, less than 356 aminoacids in length, less than 355 amino acids in length, less than 354amino acids in length, less than 353 amino acids in length, less than352 amino acids in length, less than 350 amino acids in length, lessthan 349 amino acids in length, less than 348 amino acids in length,less than 347 amino acids in length, less than 346 amino acids inlength, less than 345 amino acids in length. An OmpA protein can be 346amino acids in length. An A. baumannii OmpA amino acid sequence usefulin the compositions and methods of the invention is substantiallysimilar to the sequences set forth in Table 4 and can either be isolatedor recombinantly prepared (rOmpA). An OmpA of the present invention canhave unexpectedly high immunogenicity when compared to an OmpA that isnot, for example, at least 80 percent, at least 85 percent, at least 87percent, at least 88 percent, at least 89 percent, at least 90 percent,at least 91 percent, at least 92 percent, at least 93 percent, at least94 percent, at least 95 percent identical in amino acid sequence.

TABLE 1 A. baumannii OmpA Sequences NCBI OmpA Accession.Version NumberNCBI OmpA gi Number AAR83911.1 40287452 Q6RYW5.1 75438841 CAP01862.1169152833 CAP01565.1 169152583 CAP00823.1 169151962 CAO99984.1 169151288AAM73654.1 21666310 CAP16950.1 261599880 CAP16951.1 261599781 ACA13273.1167966448 ACA13272.1 167966446 ABY47586.1 163866832 ABY47585.1 163866830ABY47584.1 163866828 ABY47583.1 163866826 ABY47582.1 163866824ACB12042.1 170280279 ABG77310.1 110589616 ABG77309.1 110589614ABG37059.1 109675220 ABG37058.1 109675218 ABO30516.1 129307156ABO30515.1 129307154 ADX93822.1 323519441 ADX93729.1 323519348ADX91906.1 323517525 ADX91788.1 323517407 ADX91300.1 323516919YP_001847847.1 184159508 YP_001847748.1 184159409 YP_001845964.1184157625 YP_001845831.1 184157492 YP_001845494.1 184157155ZP_07242161.1 301597153 ACJ58603.1 213988304 ACJ58458.1 213988159ACJ58275.1 213987976 ACJ58171.1 213987872 ACJ56927.1 213986628ADX04874.1 322509420 ADX04775.1 322509321 ADX03387.1 322507933ADX03261.1 322507807 ADX02506.1 322507052 ZP_07242881.1 301597873ZP_07240024.1 301595016 ZP_07238125.1 301512888 ZP_07237966.1 301512729ZP_07237394.1 301512157 ZP_07227965.1 301347224 ZP_07226657.1 301345916ZP_07226582.1 301345841 ZP_07226176.1 301345435 ZP_06798301.1 294860532ZP_06797604.1 294859835 ZP_06796576.1 294858807 ZP_06795340.1 294857571ZP_06794866.1 294857097 ZP_06787675.1 294842992 ZP_06786321.1 294841638ZP_06785798.1 294841115 ZP_06785735.1 294841052 ZP_06785088.1 294840405ZP_06784877.1 294840194 ZP_06784301.1 294839618 ZP_06783732.1 294839049ZP_06783190.1 294838507 ZP_06781529.1 294836846 ZP_04663447.1 239504137ZP_04662491.1 239503181 ACC58500.1 183211102 ACC58401.1 183211003ACC56617.1 183209219 ACC56484.1 183209086 ACC56147.1 183208749 A3M8K2.2148839593 YP_001714728.1 169796935 YP_001714391.1 169796598YP_001714238.1 169796445 YP_001712610.1 169794817 YP_001712475.1169794682 YP_001707777.1 169634041 YP_001707527.1 169633791YP_001706906.1 169633170 YP_001706232.1 169632496 ABO13390.2 193078408ABO13246.2 193078282 ABO11733.2 193076988 ABO11623.2 193076900ABO11316.1 126386818 CAM87753.1 169149862 CAM87414.1 169149525CAM87256.1 169149372 CAM85607.1 169147744 CAM85470.1 169147609ZP_07237827.1 301512590 YP_002326628.1 215484397 YP_002326284.1215484059 YP_002326132.1 215483907 YP_002324545.1 215482363YP_002324452.1 215482270 YP_002320744.1 213157946 YP_002320655.1213157857 YP_002318323.1 213156662 YP_002318864.1 213156444YP_001085992.1 126643008 YP_001085848.1 126642864 YP_001084335.1126641351 YP_001084225.1 126641241 YP_001083918.1 126640934 ACJ42008.1213057106 ACJ41919.1 213057017 ACJ40724.1 213055822 ACJ40506.1 213055604ZP_07240179.1 301595171 ZP_07235999.1 301510762 ZP_07225482.1 301344741ZP_05830321.1 260558111 ZP_05829775.1 260557560 ZP_05829399.1 260557183ZP_05827995.1 260555775 ZP_05827733.1 260555512 ACA09703.1 167888787EEX05351.1 260412054 EEX03984.1 260410686 EEX02591.1 260409289EEX02489.1 260409186 EEX01692.1 260408384

The present invention provides an antigenic composition comprising atleast one antigen, wherein said at least one antigen comprises at leastpart of a protein or polypeptide of A. baumannii OmpA and comprises atleast one antigenic epitope or antigenic determinant of A. baumanniiOmpA. In one embodiment of the invention, the antigenic compositioncomprises at least one antigen that is recombinantly produced. It isfurther contemplated that the antigenic composition comprises at leastone antigen that is an isolated or purified antigen. In a furtherembodiment of the invention, the antigenic composition comprises atleast one recombinant vector and at least one polynucleotide insertedtherein that encodes said at least one protein or polypeptide, whereinthe vector is able to express said polypeptide in vivo in a mammaliansubject susceptible to infection with A. baumannii. The antigenic A.baumannii OmpA composition of the invention can be an immunogeniccomposition.

In a particular embodiment, the invention provides an isolatedpolypeptide comprising an amino acid sequence selected from SEQ IDNOS:1-6. Such polypeptides are useful in compositions of the invention,for example, pharmaceutical compositions and/or vaccine compositions.Such a vaccine composition can further comprise an adjuvant.

In another embodiment, the invention provides a composition comprisingan antigenic fragment of an amino acid sequence selected from SEQ IDNOS:1-6, wherein the antigenic fragment comprises an amino acid sequencethat differs from at least one amino acid of the amino acid sequence ofSEQ ID NO:11, or wherein the antigenic fragment comprises an amino acidsequence selected from SEQ ID NOS:7-10 and amino acids 1-18, 19-25,26-32, 51-65, 91-130, 151-153, 154-165, 166, 221-235, 265-280 and307-331 of SEQ ID NOS:1-6 (see Examples and FIG. 13). In a furtherembodiment, the composition contains an antigenic fragment that has atleast one amino acid that differs from the sequence of SEQ ID NO:11 atamino acids 35F, 39N, 48M, 56T, 83I, 85V, 119A, 124A, 128V, 129F, 131G,137V, 141M, 151E, 153E, 156P, 179I, 184A, 191G, 194H, 296A and 339N (seeFIG. 13). Such antigenic fragments can be, for example, less than 360amino acids in length less than 359 amino acids in length, less than 358amino acids in length, less than 357 amino acids in length, less than356 amino acids in length, less than 355 amino acids in length, lessthan 354 amino acids in length, less than 353 amino acids in length,less than 352 amino acids in length, less than 350 amino acids inlength, less than 349 amino acids in length, less than 348 amino acidsin length, less than 347 amino acids in length, less than 346 aminoacids in length, less than 345 amino acids in length. In addition, theantigenic fragments can be, for example, less than 340 amino acids inlength, less than 335 amino acids in length, less than 330 amino acidsin length, less than 325 amino acids in length, less than 320 aminoacids in length, less than 315 amino acids in length, less than 310amino acids in length, less than 305 amino acids in length, less than300 amino acids in length, less than 295 amino acids in length, lessthan 290 amino acids in length, less than 285 amino acids in length,less than 280 amino acids in length, less than 275 amino acids inlength, less than 270 amino acids in length, less than 265 amino acidsin length, less than 260 amino acids in length, less than 255 aminoacids in length, less than 250 amino acids in length, less than 245amino acids in length, less than 240 amino acids in length, less than235 amino acids in length, less than 230 amino acids in length, lessthan 225 amino acids in length, less than 220 amino acids in length,less than 215 amino acids in length, less than 210 amino acids inlength, less than 205 amino acids in length, less than 200 amino acidsin length, less than 195 amino acids in length, less than 190 aminoacids in length, less than 185 amino acids in length, less than 180amino acids in length, less than 175 amino acids in length, less than170 amino acids in length, less than 165 amino acids in length, lessthan 160 amino acids in length, less than 155 amino acids in length,less than 150 amino acids in length, less than 145 amino acids inlength, less than 140 amino acids in length, less than 135 amino acidsin length, less than 130 amino acids in length, less than 125 aminoacids in length, less than 120 amino acids in length, less than 115amino acids in length, less than 110 amino acids in length, less than105 amino acids in length, less than 100 amino acids in length, lessthan 95 amino acids in length, less than 90 amino acids in length, lessthan 85 amino acids in length, less than 80 amino acids in length, lessthan 75 amino acids in length, less than 70 amino acids in length, lessthan 65 amino acids in length, less than 60 amino acids in length, lessthan 55 amino acids in length, less than 50 amino acids in length, lessthan 45 amino acids in length, less than 40 amino acids in length, lessthan 35 amino acids in length, less than 30 amino acids in length, lessthan 25 amino acids in length, less than 20 amino acids in length, orless than 15 amino acids in length.

The invention further provides an isolated nucleic acid moleculeencoding an amino acid sequence selected from SEQ ID NOS:1-6 as well ascompositions comprising such nucleic acid molecules. The inventionadditionally provides a vector comprising the isolated nucleic acidmolecules of the invention. The invention also provides vaccinecomposition comprising the nucleic acid composition of the invention ora vector containing the nucleic acid molecules of the invention.

The invention further provides a composition comprising a nucleic acidmolecule encoding an antigenic fragment of an amino acid sequenceselected from SEQ ID NOS:1-6, wherein the antigenic fragment comprisesan amino acid sequence that differs from at least one amino acid of theamino acid sequence of SEQ ID NO:11, or wherein the antigenic fragmentcomprises an amino acid sequence selected from SEQ ID NOS:7-10 and aminoacids 1-18, 19-25, 26-32, 51-65, 91-130, 151-153, 154-165, 166, 221-235,265-280 and 307-331 of SEQ ID NOS:1-6. In a particular embodiment, sucha nucleic acid composition can encode an amino acid sequence, whereinthe at least one amino acid differs from the sequence of SEQ ID NO:11 atamino acids 35F, 39N, 48M, 56T, 83I, 85V, 119A, 124A, 128V, 129F, 131G,137V, 141M, 151E, 153E, 156P, 179I, 184A, 191G, 194H, 296A and 339N.

An “antigenic fragment,” “antigenic epitope” or “antigenic determinant”of A. baumannii OmpA refers to a portion of A. baumannii OmpA thateither includes or corresponds to a sequential or conformationalimmunologically active region that is recognized and bound bylymphocytes or secreted antibodies. An antigenic fragment can be anyportion up to full length of A. baumannii OmpA, for example, at leastbetween 300 to 350 amino acids, at least between 250 to 300 amino acids,at least between 200 to 250 amino acids, at least between 150 to 200amino acids, at least between 100 to 150 amino acids, at least between50 to 100 amino acids, at least between 20 to 50 amino acids, at leastbetween 10 to 20 amino acids, at least between 2 to 10 amino acids, atleast between 4 to 8 amino acids, at least between 5 to 7 amino acids.

In a further embodiment, the invention provides a vaccine compositionfor protecting a mammalian subject against infection of A. baumanniiOmpA that comprises an A. baumannii OmpA or antigenic fragment thereof,as described herein as immunizing component, and a pharmaceuticallyacceptable carrier. The vaccine compositions of the invention comprisedetoxified A. baumannii OmpA or antigenic fragment thereof that aresubstantially free of endotoxin. In certain embodiments, the vaccinecomposition can further include an adjuvant, for example, aluminiumhydroxide (AL(OH)₃) or other aluminum-containing adjuvant. Hem, S. L.and HogenEsch, H. (2006) Aluminum-Containing Adjuvants: Properties,Formulation, and Use, in Vaccine Adjuvants and Delivery Systems (ed M.Singh), John Wiley & Sons, Inc., Hoboken, N.J., USA. doi:10.1002/9780470134931.ch4. Methods for selecting an appropriate adjuvantare well known in the art and described, for example, in VaccineAdjuvants and Delivery Systems (ed M. Singh), John Wiley & Sons, Inc.,Hoboken, N.J., USA. doi: 10.1002/9780470134931.

The vaccine composition provided by the invention protects susceptiblemammals, preferably human subjects, against one or more manifestationsof A. baumannii infection, for example, blood stream infection, hospitaland community-acquired pneumonia, kidney infection, urinary tractinfection, bladder infection, wound infection, meningitis, endocarditis,endopthalmitis, and keratitis caused by A. baumannii. In someembodiments, the susceptible human subject is afflicted with diabetes,hypertension, liver cirrhosis, renal insufficiency, human immunovirusinfection, neutropenia (absolute neutrophil count more than 500cells/mm), malignancy, decubitus ulcers, septic shock, and anoxicencephalopathy; undergoing dialysis or immunosuppressive treatment; is atransplant recipient or tracheostomy patient, uses a mechanicalventilator. The vaccine composition of the invention can be particularlyindicated for active vaccination of hospital patients to preventinfections and military personnel as A. baumannii is one of the mostcommon causes for wound infection.

The vaccine composition of the invention can be provided in aphysiologically administrable form, and suitably is administrable bysubcutaneous or intranasal inoculation.

The present invention, in additional embodiments, also provides a methodfor producing an antigen or an immunogen of an antigenic composition.The method comprises (a) providing a DNA fragment encoding said antigenand introducing said fragment into an expression vector; (b) introducingsaid vector, which contains said DNA fragment, into a compatible hostcell; (c) culturing said host cell provided in step (b) under conditionsrequired for expression of the product encoded by said DNA fragment; and(d) isolating the expressed product from the cultured host cell, and,optionally, (e) purifying the isolated product from step (d) by affinitychromatography or other chromatographic methods known in the art.

In a further embodiment, the invention provides a method for preparationof a vaccine composition that contains as immunizing component, anantigenic or immunogenic composition of the invention. The methodcomprises mixing an antigenic or immunogenic composition and apharmaceutically acceptable carrier. Also provided is a method for theproduction of an antiserum that includes administering an antigenicpreparation of the invention to a mammalian host to produce antibodiesin the host and recovering antiserum containing the antibodies producedin the host. Also provided is a method of prophylactic or therapeutictreatment of A. baumannii infection in mammalian subject, suitablyhuman, comprising administering to the subject an immunologicallyeffective amount of a vaccine composition or antiserum of the inventionas described herein. In a further embodiment, the invention provides amethod for protecting a mammalian subject against A. baumanniiinfection, or reducing the severity of the infection, which comprisesinoculating the subject subcutaneously or intranasally with a vaccinecomposition of the invention to induce an immune response against A.baumannii in the subject.

The invention also provides an antibody preparation for passiveimmunization comprising at least one antibody, or antigen-bindingfragment hereof, specific for an A. baumannii OmpA protein orpolypeptide of the invention. The antibody preparation can be usedprophylactically or therapeutically against an A. baumanni infection andcan further provide passive immunization when administered to amammalian subject susceptible to infection by A. baumannii. The passiveimmunization can be an adjunct therapy to other treatments, includingactive immunization.

In a particular embodiment, the invention provides a compositioncomprising an antibody, or antigen binding fragment thereof, wherein theantibody or antigen binding fragment specifically binds to an epitopeencoded by an amino acid sequence selected from SEQ ID NOS:1-6. In afurther embodiment, the epitope can comprise an antigenic fragmentcomprising an amino acid sequence that differs from at least one aminoacid of the amino acid sequence of SEQ ID NO:11, or wherein theantigenic fragment comprises an amino acid sequence selected from SEQ IDNOS:7-10 and amino acids 1-18, 19-25, 26-32, 51-65, 91-130, 151-153,154-165, 166, 221-235, 265-280 and 307-331 of SEQ ID NOS:1-6. Forexample, the at least one amino acid can differ from the sequence of SEQID NO:11 at amino acids 35F, 39N, 48M, 56T, 83I, 85V, 119A, 124A, 128V,129F, 131G, 137V, 141M, 151E, 153E, 156P, 179I, 184A, 191G, 194H, 296Aand 339N.

The amount of vaccine of the invention to be administered a human oranimal and the regime of administration can be determined in accordancewith standard techniques well known to those of ordinary skill in thepharmaceutical and veterinary arts taking into consideration suchfactors as the particular antigen, the adjuvant (if present), the age,sex, weight, species and condition of the particular animal or patient,and the route of administration. In the present invention, the amount ofpolysaccharide-protein carrier to provide an efficacious dose forvaccination against N. meningitidis can be from between about 0.02 μg toabout 5 μg per kg body weight. In a preferred composition and method ofthe present invention the dosage is between about 0.1 μg to 3 μg per kgof body weight. For example, an efficacious dosage will require lessantibody if the post-infection time elapsed is less since there is lesstime for the bacteria to proliferate. In like manner an efficaciousdosage will depend on the bacterial load at the time of diagnosis.Multiple injections administered over a period of days can be consideredfor therapeutic usage. The compositions of the present invention can beadministered as a single dose or in a series (i.e., with a “booster” or“boosters”). In one embodiment of the invention, a preferred route ofadministration is intramuscular or subcutaneous, with intramuscularroute preferred. Administration can be by injection or by an alternativedelivery device.

In a preferred embodiment of the invention, the vaccine composition isformulated as a sterile liquid, pyrogen-free, phosphate-bufferedphysiological saline, with or without a preservative. The choice ofsuitable carriers and other additives will depend on the exact route ofadministration and the nature of the particular dosage form, e.g.,liquid dosage for (e.g., whether the composition is to be formulatedinto a solution, a suspension, gel or another liquid form), or soliddosage form (e.g., whether the composition is to be formulated into apill, tablet, capsule, caplet, time release form or liquid-filled form).

An antibody of the invention, or a fragment thereof, specifically bindsto A. baumannii OmpA and is well tolerated by the human immune system.

An antibody refers to a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive, antigen-binding portion of an immunoglobulin molecule, like anantibody fragment. As described in more detail below, an antibodyfragment is a portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab,Fv, scFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the intact antibody.The term antibody fragment also includes isolated fragments consistingof the variable regions, such as the “Fv” fragments consisting of thevariable regions of the heavy and light chains and recombinant singlechain polypeptide molecules in which light and heavy variable regionsare connected by a peptide linker (“scFv proteins”). As used herein, theterm antibody fragment does not include portions of antibodies withoutantigen binding activity, such as Fc fragments or single amino acidresidues. Other antibody fragments, for example single domain antibodyfragments, are known in the art and can be used in the claimedconstructs. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yauet al., J Immunol Methods 281:161-75 (2003); Maass et al., J ImmunolMethods 324:13-25 (2007); Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y. (1988)).

In one embodiment, the invention provides an antibody, or fragmentthereof, that selectively binds to A. baumannii OmpA, or an antigenicfragment thereof and is humanized or fully human. The antibody, orfragment thereof, displays a high affinity for A. baumannii OmpA, or anantigenic fragment thereof. The present invention therefore relates tomonoclonal or polyclonal antibodies, and fragments thereof, which bindspecifically to an A. baumannii OmpA, or an antigenic fragment thereof.

The antibody of the invention, or fragment thereof, is preferably chosenso that it has particular binding kinetics (e.g. high affinity, littledissociation, low off rate, strong neutralizing activity) for thespecific binding to A. baumannii OmpA, or an antigenic fragment thereof.The antibodies are preferably isolated antibodies. According to afurther aspect, the antibodies are neutralizing antibodies. Theantibodies of the invention include in particular monoclonal andrecombinant antibodies. A monoclonal antibody of the invention isderived from a hybridoma (e.g. an antibody which is secreted by ahybridoma produced by means of hybridoma technology such as thestandardized hybridoma methods of Miller and Milstein). An antibody ofthe invention can be derived from a hybridoma and have specificity foran A. baumannii OmpA, or an antigenic fragment thereof.

The antibodies of the invention can comprise an amino acid sequence thatderives completely from a single species, and thus can be for example ahuman antibody or a mouse antibody. According to further embodiments,the antibody can be a chimeric antibody or a CDR graft antibody oranother type of humanized antibody.

The term “antibody” is intended to refer to immunoglobulin moleculesthat are formed of four polypeptide chains, two heavy (H) chains and twolight (L) chains. The chains are usually linked together by disulfidebonds. Every heavy chain is composed of a variable region of the heavychain (abbreviated here to HCVR or VH) and a constant region of theheavy chain. The constant region of the heavy chain is formed from threedomains CH1, CH2 and CH3. Each light chain is composed of a variableregion of the light chain (abbreviated here to LCVR or VL) and aconstant region of the light chain. The constant region of the lightchain is formed from a CL domain. The VH and VL regions may be furtherdivided into hypervariable regions which are referred to ascomplementarity-determining regions (CDR) and are interspersed with moreconserved regions which are referred to as framework regions (FR). EachVH and VL region is formed from three CDRs and four FRs which arearranged from the N terminus to the C terminus in the followingsequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term “fragment” or “antigen-binding fragment” or “binding fragment”used in reference to an antibody refers to one or more fragments of anantibody having specificity for an A. baumannii OmpA, the fragment(s)still having the ability to bind specifically the A. baumannii OmpA, oran antigenic fragment thereof. It has been shown that theantigen-binding function of an antibody can be undertaken by fragmentsof a complete antibody. Examples of binding fragments include anantibody (i) an Fab fragment, i.e. a monovalent fragment composed of theVL, VH, CL and CH1 domains; (ii) an F(ab).sub.2 fragment, i.e. abivalent fragment which comprises two Fab fragments linked together by adisulfide bridge in the hinge region; (iii) an Fd fragment which iscomposed of the VH and CH1 domains; (iv) an Fv fragment which iscomposed of the VL and VH domains of a single arm of an antibody; (v) adAb fragment (Ward et al., (1989) Nature 341:544-546) which consists ofa VH domain or VH, CH1, CH2, DH3, or VH, CH2, CH3; and (vi) an isolatedcomplementarity-determining region (CDR). Although the two domains ofthe Fv fragment, namely VL and VH, are encoded by separate genes theycan furthermore be connected together by a synthetic linker by use ofrecombinant methods, whereby they can be produced as a single proteinchain in which the VL and VH regions are present together in order toform monovalent molecules (known as single-chain Fv (ScFv), see, forexample, Bird et al., Science 242:423-426 (1988); and Huston et al.,Proc. Natl. Acad. Sci. USA 85:5879-5883 ((1988). Such single-chainantibodies are also intended to be encompassed by the term “antigenicfragment” of an antibody. Other types of single-chain antibodies such asdiabodies likewise belong thereto. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but with use of a linker that is too short for thetwo domains to be present together on the same chain, the domains thusbeing forced to pair with complementary domains of another chain and toform two antigen-binding sites (see, for example, Holliger, P., et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak, R. J., et al.,Structure 2:1121-1123 (1994).

A further embodiment is for an antibody or antigen-binding fragmentthereof to be part of a larger immunoadhesion molecule which is formedby covalent or non-covalent association of the antibody or antibody partwith one or more further proteins or peptides. Such immunoadhesionmolecules can involve the use of the streptavidin core region in orderto produce a tetrameric scFv molecule (Kipriyanov, S. M., et al. HumanAntibodies and Hybridomas 6:93-101 (1995) and the use of a cysteineresidue, of a marker peptide and of a C-terminal polyhistidine tag inorder to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al., Mol Immunol 31:1047-1058 (1994).

Antibody parts, such as Fab and F(ab′)₂ fragments, can be produced fromwhole antibodies by using conventional techniques such as digestion withpapain or pepsin. It is additionally possible to obtain antibodies,antibody parts and immunoadhesion molecules by using standardizedrecombinant DNA techniques.

An antibody specific to A. baumannii OmpA, or an antigen-bindingfragment thereof can be produced, expressed, generated or isolated byusing recombinant techniques, such as antibodies which are expressed byuse of a recombinant expression vector transfected into a host cell;antibodies isolated from a recombinant combinatorial antibody library;antibodies isolated from an animal (e.g. a mouse) which is transgenicdue to human immunoglobulin genes (see, for example, Taylor, L. D., etal., Nucl Acids Res. 20:6287-6295 (1992); or antibodies which areproduced, expressed, generated or isolated in any other way in whichparticular immunoglobulin gene sequences (such as human immunoglobulingene sequences) are combined with other DNA sequences. Recombinantantibodies include, for example, chimeric, CDR graft and humanizedantibodies.

A human antibody that has specificity for an A. baumannii OmpA hasvariable and constant regions corresponding to immunoglobulin sequencesof the human germline, as described for example by Kabat et al. (seeKabat, et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242), or is derived therefrom. The human antibodiesof the invention can, however, comprise amino acid residues which arenot encoded by human germline immunoglobulin sequences (e.g. mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs, and especially in CDR3.Recombinant human antibodies of the invention have variable regions andcan also comprise constant regions derived from immunoglobulin sequencesof the human germline (see Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). According toparticular embodiments, such recombinant human antibodies are, however,subjected to an in vitro mutagenesis (or to a somatic in vivomutagenesis if an animal which is transgenic due to human Ig sequencesis used), so that the amino acid sequences of the VH and VL regions ofthe recombinant antibodies are sequences which, although they arerelated to VH and VL sequences of the human germline or are derivedtherefrom, do not naturally exist within the human antibody germlinerepertoire in vivo. According to particular embodiments, suchrecombinant antibodies are the result of a selective mutagenesis orback-mutation, or both.

In a further embodiment, the invention provides methods of diagnosis ofA. baumannii infection comprising obtaining a tissue sample from asubject suspected of A. baumannii infection, contacting the tissuesample suspected of comprising A. baumannii with an OmpA fragment,primer, antibody or antigen-binding fragment thereof and detecting thepresence of A. baumannii OmpA in the sample by methods known in the art.

The invention will be further described by reference to the followingillustrative, non-limiting examples setting forth in detail severalpreferred embodiments of the inventive concept. Other examples of thisinvention will be apparent to those skilled in the art without departingfrom the spirit of the invention.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

Example I Specific Anti-A. baumannii Antibodies are Generated DuringInfection in Mice

Six clinical isolates of A. baumannii were used (Table 2). Theseisolates were harvested from various body sites of infection. Five ofthe strains were resistant to all antibiotics except for colistin (Table5). Strain typing was performed by multi-locus sequence typing aspreviously described (Bartual et al., J Clin Microbiol 43:4382-4390(2005); Tian et al., Antimicrob Agents Chemother 55:429-432 (2011).Balb/c mice were used for all experiments. For some experiments, retiredbreeder mice (>6 mo old) were used, whereas for other experimentsjuvenile (6-10 weeks old) Balb/c mice were used. Diabetes was induced byintraperitoneal injection of 200 mg/kg streptozotocin in 0.2 ml citratebuffer 10 days prior to infection. Glycosuria and ketonuria wereconfirmed in all mice 7 days after streptozotocin treatment, aspreviously described (Ibrahim et al., J Antimicrob Chemother58:1070-1073 (2006); Ibrahim et al., J Clin Invest 117:2649-2657 (2007);Spellberg et al., Antimicrob Agents Chemother 49:830-832 (2005).Bacterial strains used are described in Table 2.

A. baumannii cell membrane preparations were produced by a modificationof a standard, published method (Molloy et al., Eur J Biochem267:2871-2881 (2000); Soares et al., Proteome Sci 7:37 2009). In brief,A. baumannii strains were grown overnight at 37° C. with shaking intryptic soy broth (TSB). The bacteria were passaged to mid-log-growth at37° C. with shaking. The cells were harvested by centrifugation at 3,500g for 15 min at 4° C. and washed twice with 10 mL 0.9% (w/v) NaCl. Theresultant pellet was resuspended in disintegration buffer (7.8 g/LNaH₂PO₄, 7.1 g/L Na₂HPO₄, 0.247 g/L MgSO4 7.H₂O+protease inhibitor mix(GE Healthcare, USA)+nuclease mix (GE Healthcare, USA)) and sonicated onice for 3 periods of 5 min. The unbroken cells were separated bycentrifugation at 1,500 g. The supernatant was centrifuged for 30 min at4° C. at 4,500 rpm and was passed through a 0.45 μM filter (Milipore,USA) to remove cell debris. An equal volume of ice-cold 0.1 M sodiumcarbonate (pH 11) was added to the resulting supernatant and the mixturewas stirred slowly overnight, on ice. The carbonate treated membraneproteins were collected by ultracentrifugation at 100,000 g for 45 minat 4° C., and the membranes were re-suspended in 500 μl H₂O. Finally,the protein extract was processed with a 2-DE Cleanup Kit (Bio-Rad,USA).

Two dimensional SDS/10%-PAGE gels of A. baumannii cell membranepreparations were used to separate proteins by size and isoelectricfocusing (IEF), as described by Pitarch et al (Pitarch et al., Mol CellProteomics 5:79-96 (2006); Pitarch et al., Electrophoresis 20:1001-1010(1999). For isoelectric focusing (IEF), the Bio-Rad-PROTEIN IEF systemwas used (Bio-Rad, USA) with 4-7 pH gradient strips (ReadyStrip IPGstrips, Bio-Rad, USA). Proteins were solubilized in 8 M urea, 2% (w/v)CHAPS, 40 mM DTT and 0.5% (v/v) corresponding rehydrated buffer(Bio-Rad, USA). The strips were rehydrated overnight and underwentelectrophoresis at 250 V for 20 min, 4000 V for 2 h, and 4,000 V for10,000 V-h, all at room temperature. Prior to the second dimension(SDS-PAGE), the focused IPG strips were equilibrated with buffer I andII for 10 min (ReadyPrep 2-D Starter Kit, Bio-Rad, USA). The proteinswere separated on 8-16% Criterion Pre-cast Gel (Bio-Rad, USA) andtransferred to immune-Blot PVDF membranes (Bio-Rad, USA). Membranes weretreated with Western Blocking Reagent (Roche) overnight and probed withpre-immune or immune A. baumannii infected-mice serum. Membranes werewashed and incubated with secondary, HRP-conjugated goat anti-mouse IgG(Santa Cruz Biotech, USA). After incubation with SuperSignal West DuraExtended Duration Substrate (Pierce, USA), signals were detected using aCCD camera.

Protein spots of interest were excised and sent to the UCLA W. M. KeckProteomic Center for identification on a Thermo LTQ-Orbitrap XL massspectrometer (San Jose, Calif.) equipped with an Eksigent (Dublin,Calif.) NanoLiquid chromatography-1D plus system and an Eksigentautosampler. Proteins within the spots were in-gel tryptic digested asdescribed by Shevchenko et al. (Shevchenko et al., Proc Natl Acad SciUSA 93:14440-14445 (1996); Shevchenko et al., Anal Chem 68:850-858(1996). The eluted peptides were loaded onto a CVC Microtech (Fontana,Calif.) 35 mm length, 100 μm ID C18 pre-Trap column and washed for 10min with 100% Buffer A (2% acetonitrile containing 0.1% formic acid) ata flow rate of 5 μl/min. The peptides were separated on a 15 cm NewObjective ProteoPep IntegraFrit column (Woburn, Mass.) using a flow rateof 300 nl/min. The following elution gradient was used: 0-15 min 0-30%Buffer B (98% acetonitrile containing 0.1% formic acid), 15-20 min30-80% Buffer B and 20-22 min 80% Buffer B. The column was thenre-equilibrated for 13 min with Buffer A. The eluting analytes weresprayed in positive mode into the LTQ-Orbitrap MS using electrosprayionization voltage of 2300 V, capillary voltage of 45 V, tube lens of130 V, and capillary temperature of 200° C. Information dependentacquisition was performed where the 6 most intense ions were selected inthe m/z range of 300-1600 using a 60 K resolution FTMS scan andsubjecting them to MS-MS using broadband collision induceddisassociation of normalized collision energy of 35 and LTQ detection.Peaks were excluded from further MS-MS for a period of 60 sec.

The resulting MS/MS spectra was searched against the Acinetobacterbaumannii strain ATCC 17978 database(gib.genes.nig.ac.jp/single/blast2/main.php?spid=Abau_ATCC17978) usingthe Matrix Science MASCOT Daemon search engine (Boston, Mass.). Thefollowing search parameters were used: peptide tolerance: ±10 ppm, MS/MStolerance ±0.3 Da, maximum missed cleavages: 2, fixed modifications:carboxymethyl (C) and variable modifications: deamidization (ND) andoxidation (M). Proteins identified within a particular included thosewith a minimum of two unique peptides that are ranked as number 1 andwith an ion scores with a p<0.05.

His-tagged rOmpA (amino acids 2 to 347) was produced in an Escherichiacoli pQE-32 expression system (Qiagen) as previous described (Luo etal., J Infect Dis 201:1718-1728 (2010); Spellberg et al., Infect Immun76:4574-4580 (2008). Briefly, ompA was amplified from A. baumannii 17978genomic DNA with primers:

OmpA-F CATCACCATGGGATCCTTGTTGCTGCTCCATTAGCT andOmpA-R CTAATTAAGCTTGGCTGCAGTTATTGAGCTGCTGCAGGAand cloned into BamHII and Pst I sites of QE-32 by using In-Fusion 2.0Dry-Down PCR Cloning Kit, per the manufacturer's instructions (ClontechLaboratories). The 6X-His tagged protein was purified over a Ni-agaroseaffinity column according to the manufacturer instructions (Qiagen).Endotoxin was removed from rOmpA by using Detoxin Gel Endotoxin RemovingColumns (Norgen Biotek, Canada), and the endotoxin level was determinedwith Limulus Amebocyte Lysate endochrome (Charles River) permanufacturer's instruction. Using this procedure, endotoxin was reducedto <1EU per dose used for vaccination. Mice were immunized bysubcutaneous injection of rOmpA in 0.1% Al(OH)₃ (Alhydrogel, BrenntagBiosector, Frederikssund, Denmark) in phosphate buffered saline (PBS).Control mice received adjuvant alone on the same schedule. Mice wereimmunized 5 weeks prior to infection and again 2 weeks prior toinfection. Four days after the boost (10 days prior to infection), micewere rendered diabetic as described above.

A. baumannii strains were grown overnight at 37° C. with shaking in TSBbroth. The bacteria were passaged to mid-log-growth at 37° C. withshaking Cells were washed twice with PBS and resuspended at theappropriate concentration for infection. The final concentration wasconfirmed by quantitative culturing of the inocula. Mice were infectediv via the tail-vein with sublethal (10⁶) or lethal (targeted 2×10⁷)inocula in PBS. All animal experiments were approved by theInstitutional Committee on the Use and Care of Animals at the LosAngeles Biomedical Research Institute.

Two days after infection (the day on which control mice were anticipatedto begin dying), organs were harvested and homogenized in sterile PBSwith 1% triton with protease inhibitor cocktail (Sigma-Aldrich Corp. St.Louis, Mo., USA). Homogenized organs from individually marked mice werequantitatively cultured to determine tissue bacterial burden.

A previously published ELISA assay (Ibrahim et al., Infect Immun74:3039-3041 (2006); Ibrahim et al., Infect Immun 73:999-1005 (2005);Spellberg et al., J Infect Dis 194:256-260 (2006); Spellberg et al.,Infect Immun 73:6191-6193 (2005) was adapted for detection of antibodiesagainst A. baumannii cell membrane preparations and rOmpA. In brief,ELISA plates were coated with 100 μl per well of 5 μg/ml of rOmpA orcell membrane preparation. Coated wells were blocked with bovine serumalbumin, incubated with mouse sera, washed, and stained with goatanti-mouse secondary antibody conjugated with horseradish peroxidase.Wells were washed again and incubated with o-phenylenediamine substratewith H₂O₂. The color was allowed to develop for 20 min after which thereaction was terminated by adding equal volume of 3N HCl and the opticaldensity (OD) was determined at 490 nm in a microtiter plate reader.Negative control wells received an irrelevant isotype control monoclonalantibody rather than mouse serum. The ELISA titer was taken as thereciprocal of the last serum dilution with an OD reading≧(mean OD ofnegative control samples+(standard deviation*2)).

A. baumannii HUMC1 was cultured overnight in tryptic soy broth (TSB) at37° C., passaged to mid-log growth, rinsed, and aliquoted into 96 wellmicrotiter plates. For complement studies, non-immune or immune serawere added to the wells for 1 hour. Well contents were quantitativelycultured at baseline and again at 1 h. The opsonophagocytic kill assaywas based on a modification of a previously used method [25-26]. MurineRAW 264.7 macrophage cells (both from American Type Culture Collection,Rockville, Md.) were tested because they are known to be capable ofkilling microbes after differentiation [15-17]. The cells were culturedat 37° C. in 5% CO₂ in RPMI 1640 (Irvine Scientific, Santa Ana, Calif.)with 10% fetal bovine serum (FBS), 1% penicillin, streptomycin, andglutamine (Gemini BioProducts), and 50 μM β-mercaptoethanol(Sigma-Aldrich, St. Louis, Mo.). RAW 274.7 cells were activated by 3days of exposure to 100 nM PMA (Sigma-Aldrich). Activated RAW 264.7macrophages were harvested after scraping with BD Falcon cell scrapers(Fischer Scientific) and added to the microtiter wells at a 20:1 ratioof macrophages to bacteria. After a 1 hour incubation with gentleshaking, aliquots from the wells were quantitatively plated in trypticsoy agar (TSA). Colony forming units (CFU) of the co-cultured tubes werecompared to CFUs of growth control tubes containing only microbes withno macrophages. Percent killing was calculated as 1−(CFUs fromco-culture wells/CFUs from growth control wells without macrophages).

Survival was compared by the non-parametric Log Rank test. Antibodytiters, bacterial burden, MPO levels, and cytokine levels were comparedwith the Wilcoxon Rank Sum test for unpaired comparisons or the WilcoxonSigned Rank test for paired comparisons, as appropriate. Correlationswere determined by the Spearman Rank test. All statistics were run usingKyplot. Differences were considered significant if the p value was<0.05.

As a basis for identifying lead antigenic candidates for vaccinedevelopment, the humoral immune response to surface proteins from A.baumannii was determined after natural infection. Since diabetes is arisk factor for acquisition of and worse outcomes from A. baumanniiinfection (Alsultan et al., J Chemother 21:290-295 (2009); Furniss etal., J Burn Care Rehabil 26:405-408 (2005); Metan et al., Eur J InternMed 20:540-544 (2009), a diabetic ketoacidosis (DKA) mouse model ofmucormycosis (Ibrahim et al., J Antimicrob Chemother 58:1070-1073(2006); Ibrahim et al., J Clin Invest 117:2649-2657 (2007); Spellberg etal., Antimicrob Agents Chemother 49:830-832 (2005) was adapted for invivo study of A. baumannii infections. Individually marked mice in DKAwere bled via tail-vein nicking to determine baseline, pre-immuneanti-A. baumannii cell membrane protein antibody titers. Mice were theninfected via the tail-vein with survivable inocula of six clinicalisolates of A. baumannii (Table 2 and Table 5). Two weekspost-infection, paired immune sera were obtained from the mice. ELISA ofpaired pre-immune vs. immune sera confirmed that mice infected with allof the strains generated substantial increases (10-100-fold) in anti-A.baumannii cell membrane IgG-antibody titers by 2 weeks post-infection(FIG. 1).

Having demonstrated a specific humoral immune response to the organism,the immunodominant antigenic target of that response was sought. A.baumannii cell membrane protein preparations from all six strains usedto infect mice were separated by two dimensional gel electrophoresis andstained by western blot using paired pre-immune and immune sera from theabove infected mice. The two dimensional gels demonstrated effectiveseparation by size and isoelectric focusing (IEF) of membrane proteinsfrom all six clinical isolates (FIG. 2A). In all cases, post-immuneserum identified a limited number of unique spots not recognized bypre-immune serum (FIG. 2B).

The same three spots (FIG. 2B) were selected for identification byMALDI-TOF analysis across blots from three different A. baumanniiisolates representing different strain types (Table 2). The proteinfound in all spots was identified as OmpA, which is known to be apredominant component of the outer cell membrane of A. baumannii (Choiet al., Cell Microbiol 10:309-319 (2008). Anti-OmpA antibody titers weredetermined in paired pre-immune vs. immune sera from mice infected withA. baumannii. As for total anti-A. baumannii antibodies, anti-rOmpA IgGtiters increased in all mice infected with A. baumannii (FIG. 3),confirming that OmpA is a target of adaptive humoral immunitypost-infection.

Example II OmpA as a Vaccine Antigen

Ideal antigens for vaccine development should be conserved acrossclinical isolates and should not be homologous to the human proteome.The OmpA gene was sequenced in the six clinical isolates used forinfection. The protein sequence had 99% identity across all clinicalisolates (FIG. 4), which were harvested 58 years apart (1951 to 2009)from varied clinical sources (cerebrospinal fluid, lung, blood, wound),and included both carbapenem-resistant and a carbapenem-susceptiblestrain (Table 2 and Table 5). Alignment against 14 other sequences fromA. baumannii in PubMed revealed 89% identity across all sequences (Table4). PubMed BLAST search of the human proteome using the ATCC 17978 OmpAsequence revealed only 7 sequences with minimal homology (E valuesranging 0.53 to 6.2). Thus OmpA is conserved across a broad array ofclinical isolates of A. baumannii but shares minimal homology with humanproteins.

rOmpA was expressed in E. coli and purified by nickel-agarose binding toa His tag. Endoxotin levels were reduced to less than 1 EU per vaccinedose. In the initial experiment, retired breeder (>6 months old) micewere vaccinated and boosted with rOmpA in 0.1% aluminum hydroxide(Al(OH)₃). Two weeks after the boost, the DKA mice were infected via thetail-vein with A. baumannii HUMC1. Vaccinated mice had significantimprovements in survival compared to adjuvant control mice (FIG. 5A).The experiment was repeated using juvenile mice and with multiplevaccine doses. All vaccine doses improved survival compared to adjuvantcontrol mice, and a dose response was found with 100 μg having thegreatest efficacy, which was significantly superior to the 3 μg dose(FIG. 5B).

To determine the impact of vaccination on bacterial burden, juvenilemice were vaccinated, made diabetic, and infected as above. On day 2post-infection (the day the control mice were predicted to die based onthe previous experiment), mice were euthanized and organs harvested todetermine tissue bacterial burden. Vaccination reduced by approximately10-fold the tissue bacterial burden in all organs evaluated except forthe lungs, which had a non-significant (p=0.08) 3-fold reduction inbacterial burden (p<0.01 bacterial burden in vaccinated vs. control micefor all other organs) (FIG. 5C).

To confirm efficacy in a second animal model, an established model of A.baumannii pneumonia in rats was used (Russo et al., Infect Immun76:3577-3586 (2008); Russo et al., J Infect Dis 199:513-521 (2009). Inbrief, Long-Evans rats (250 to 300 g) were anesthetized with 3.5%halothane in 100% oxygen until unconscious and then maintained at 3.5%halothane. The trachea was exposed surgically, and a 4-in. piece of 1-0silk was slipped under the trachea to facilitate instillation of theinoculum. The animals were suspended in a supine position on a60°-incline board. Pulmonary instillation of bacteria in PBS wasintroduced intratracheally (1.2 ml/kg of body weight) via a 1-ml syringeand 26-gauge needle, and the incision was closed with surgical staples.Lungs were harvested at 24 and 48 hours, homogenized, and quantitativelycultured to determine bacterial burden. This model recapitulatesaspiration via the upper airways, which is a common mode of A. baumanniiclinical pneumonia in intensive care units, without requiring immunesuppression (Russo et al., Infect Immun 76:3577-3586 (2008). Rats werevaccinated, boosted, and infected intratracheally two weeks after theboost. Lung bacterial burden was assessed at 24 and 48 hours. (FIG. 5D).

Example III Antibodies in Vaccine-Mediated Protection

The relationship between antibody titers and survival in vaccinated micewas evaluated. Given the approximate 50% survival seen in micevaccinated with 3 μg, this dose was chosen for antibody-survivalanalysis, to enable a mixture of vaccinated mice that survived or didnot survive the infection. In two separate experiments, mice werevaccinated with 3 μg or adjuvant alone, boosted, and antibody titersdetermined pre-infection. Vaccination induced marked increases inanti-rOmpA IgG antibody titers (median [range] titers=204,800[102,400-409,600] vs. 800 [400-1,000] for vaccinated vs. control mice,p<0.0001). Because the infectious inocula were somewhat lower in theseexperiments (1.4×10⁷ and 1.6×10⁷) than in the previous (2×10⁷), morethan 50% of vaccinated mice survived despite the use of the 3 μg vaccinedose (FIG. 6A). Antibody titers correlated with survival when analyzingboth vaccinated and control mice combined (p<0.0001, rho=0.6) or justanalyzing vaccinated mice without control mice (p=0.0009, rho=0.6 bySpearman Rank test, FIG. 6B). An IgG titer threshold of ≧204,800 wasmaximally accurate (98%) at distinguishing survivors from non-survivorswhen analyzing both vaccinated and control mice, whereas titers ofeither ≧102,400 or 204,800 both had the same maximal accuracy (85%) whenjust analyzing vaccinated mice (Table 3).

The correlation of antibody titer with survival suggested thatantibodies were rOmpA vaccine effectors. B cell deficient mice wereinfected with A. baumannii HUMC1 to determine if mice deficient in thesecell types were susceptible to infection, but no deaths occurred and themice never appeared clinically ill. Furthermore, B cell deficient micewere resistant to diabetes induction, making comparisons problematicbetween B cell deficient and wild type mice. Therefore, rather thandisrupting B lymphocyte function, donor mice were vaccinated with rOmpAor adjuvant alone and immune or control serum harvested by terminalbleed. rOmpA titers in immune serum were higher than in control serum(1:409,600 vs. 1:3200). DKA mice were treated ip with 0.5 ml of immuneor control serum and infected 2 hours later with A. baumannii HUMC 1.Mice treated with immune serum had markedly enhanced survival vs. micetreated with control serum (FIG. 7A).

To define the mechanism of antibody-induced protection, A. baumannii wascultured in the presence of immune vs. non-immune serum. A. baumanniinumbers increased after 1 hour culture in both sera, excludingcomplement-mediated killing as a mechanism of protection. However,immune serum did enhance macrophage opsonophagocytic killing of A.baumannii (FIG. 7B).

TABLE 2 Bacterial Strains* Strain Carbapenem Strain Type SourceResistant? Comments ATCC ST112 ATCC; No Isolated in 1951 17978cerebrospinal from a 4 month fluid isolate old with fatal meningitis(Piechaud and Second, 1951) HUMC1 ST206 HUMC, blood Yes Bacteremic VAPand sputum isolate HUMC4 ST208 HUMC, deep Yes VAP endotracheal aspirateHUMC5 ST208 HUMC, Yes VAP bronchoalveolar lavage HUMC6 ST208 HUMC,sputum Yes VAP HUMC12 ST208 HUMC, wound Yes Infected diabetic infectionstump wound *HUMC = clinical isolates from in-patients in 2009; VAP =ventilator associated pneumonia. Susceptibility results shown in Table5.

TABLE 3 Accuracy of anti-rOmpA IgG Antibody Titer Cut Offs forPredicting Survival in Vaccinated and Control Mice Infected with A.baumannii HUMC1 Sensitivity* Specificity* PPV* NPV* Accuracy* IgG Titers≧25,600 100% (100%) 76% (0%) 71% (70%) 100% (N/A)^(†) 85% (70%) ≧51,200100% (100%) 80% (17%) 75% (74%) 100% (100%) 88% (75%) ≧102,400 100%(100%) 88% (50%) 83% (82%) 100% (100%) 93% (85%) ≧204,800  43% (86%) 96%(83%) 86% (92%)  76% (71%) 98% (85%) ≧409,600  43% (43%) 96% (100%) 86%(100%)  76% (43%) 78% (60%) Numbers shown are for all 40 vaccinated andcontrol mice, or for just the 20 vaccinated mice (in parenthesis).*Sensitivity = number of surviving mice with titers ≧ the cut-off/numberof all surviving mice; Specificity = number of mice that died withtiters < the cut-off/number of all mice that died; PPV = positivepredictive value, which is the percentage of mice with titers ≧ thecut-off that survived; NPV = negative predictive value, which is thepercentage of mice with titers < cut-off that died; Accuracy = [(numberof mice with titers ≧ the cut-off that survived infection) + (number ofmice with titers < the cut-off that died from infection)/(all mice)].^(†)No vaccinated mice had titers <25,600, so NPV cannot be calculated.

TABLE 4 Alignment

gi|148839593: ATCC 17978, gi|260557183: ATCC 19606, gi|184159409: ACICU,gi|163866826: DM511 (PMID 18591275), gi|129307154: 16B, gi|163866824:IF501 (PMID 18591275) gi|163866832: LI311 (PMID 18591275), gi|169632496:SDF, gi|213057017: AB0057, gi|169794817: AYE, gi|163866830: BD335 (PMID18591275), gi|129307156:, gi|21666310:, gi|163866828: KB167 (PMID18591275), gi|239501745: AB900

TABLE 5 Antibacterial Minimum Inhibitory Concentrations (μg/ml) forClinical Isolates Used in the Current Study. Ampicillin/ Pipercillin/Strain Amikacin Gentamicin Aztreonam sulbactam tazobactam CefepimeMeropenem ATCC 8 8 16   1/0.5   0.06/4 2 0.25 17978 HUMC1 >128 >128 6416/8   <128/4 16 32 HUMC4 >128 >128 32 32/16  <128/4 16 8HUMC5 >128 >128 32 32/16  <128/4 16 8 HUMC6 >128 >128 32 32/16  <128/416 8 HUMC12 >128 >128 32 32/16  <128/4 16 4 Strain Imipenem ErtapenemDoripenem Ciprofloxacin Tigecycline Colistin ATCC 0.25 4 0.5 0.125 0.252 17978 HUMC1 16 128 16 >128 4 2 HUMC4 4 32 4 64 4 2 HUMC5 4 32 8 64 4 2HUMC6 4 32 4 64 4 2 HUMC12 2 16 8 64 4 2

Example IV The Impact of Vaccine Dose on Immunogenicity

The impact of vaccine dose on the nature of the immune response to therOmpA vaccine was explored. Mice were vaccinated as above. Two weeksafter the boost, serum and splenocytes were harvested. Median[interquartile ranges] antibody titers for control, 3, 30, and 100 μgdose vaccinated mice were 2,400 [800-3,200], 51,200 [51,200-102,400],204,800 [102,400-204,800], and 204,800 [89,600-512,000] (p<0.001 for allvaccinated doses vs. control and <0.05 for both 30 and 100 μg dose vs. 3μg dose) (FIG. 8A).

IgM responses were substantially higher in response to the 30 and 100 μgdoses than the 3 μg dose (median titer 1:12,000 for both higher dosesvs. 1:800 for the 3 μg dose and adjuvant control mice, p<0.05) (FIG.6B). IgG1 was the predominant Ig subtype found, with median titers of1:320,000 to 1:1,600,000 for vaccinated mice vs. 1:400 for control mice(p<0.05 for all vs. control). IgG1 titers were significantly higher formice vaccinated with 100 μg than 3 μg (p=0.02). Median IgG2a and 2btiters were substantially lower than IgG1 titers but still significantlyabove the titers in control mice (FIG. 8B). IgG3 titers were much lower,with median titers of 1:800 for all three vaccinated groups, but stillsignificantly higher than control mice (median 1:200).

Similarly to antibody responses, all doses of vaccine mediatedsignificant increases in IFNγ, IL-4, and IL-17 production bysplenocytes, versus splenocytes from control mice (FIG. 9A). IL-4production was maximal at the highest (100 μg) dose of vaccine. Comparedto the baseline IFNγ-predominant IFNγ:IL-4 ratio after stimulation withcontrol (unvaccinated) splenocytes by rOmpA, all doses of vaccinesmediated more balanced ratios (median [interquartile] ratios=3.2[1.3-5.8] for control vs. 1.0 [0.8-1.3], 0.9 [0.7-1.1], and 0.5[0.5-0.7] for control vs. 3, 30, and 100 μg doses, respectively). TheTh1:Th2 ratio was significantly lower for the 100 μg dose than for allother groups (p<0.02 for all comparisons).

T cell and B cell immunodominant epitopes were defined using overlappingpeptides. Immunodominant T cell epitopes were defined as those inducingcytokine responses above the 3rd quartile across all 15mers tested. Inmice vaccinated with 3 μg, only 4, 5, and 5 peptides were found to meetthis criteria for IFN-γ, IL-4, and IL-17, respectively (FIG. 10).Distinct peptides were found to induce the three cytokines fromsplenocytes. Of interest was that peptide 1 was by the far most potentinducer of IFN-γ production and peptide 2, which overlapped with peptide1 by 5 amino acids, induced substantially more IL-4. Only 2 consensusepitopes were found to induce all three cytokines from splenocytesharvested from mice vaccinated with 3 μg (peptides 23 and 30).

Example V Epitope Mapping of Anti-OmpA Polyclonal Immune Serum

To identify B cell epitopes, immuno dot blots were conducted usingimmune serum and membranes containing overlapping peptides. In brief,overlapping 12-mer peptides, offset by five amino acids weresynthesized, covalently bound at the C terminus to a Whatman 50cellulose membrane, and directly probed with the immune serum. Themembranes were counter-stained with secondary anti-mouse IgG antibody,washed four times in T-TBS (TBS containing 0.05% Tween 20), andincubated with a 1:3,000 dilution of horseradish peroxidase-conjugatedProtein G (Bio-Rad, Hercules, Calif.) in blocking buffer. The membraneswere processed for film development (chemiluminescent detection) with anAmersham Pharmacia Biotech ECL kit (Piscataway, N.J.). TIF images weregenerated with the Bio-Rad Gel Doc 2000 Imaging System and densitometryused to define quantitative reactivity. A number of specific B cellepitopes were identified (FIG. 11). Only 3 peptides were found torepresent both B cell and T cell epitopes (2, 16, and 23). Homologymodeling revealed that the predominant B cell epitopes were localized tosurface exposed a helices and β sheets, although surprisingly there wasalso a dominant B cell epitope on the cytoplasmic face of the protein ata hairpin loop structure (FIG. 12).

rOmpA was modeled in silica by the SWISS-MODEL fully automated proteinstructure homology-modeling server accessible via the ExPASy web server.The model was optimized by energy minimization using Discovery Studioversion 2.1 (Accelrys, San Diego, Calif.). The minimization wasperformed in several steps, using a steepest descendent and conjugategradient algorithm to reach the minimum convergence (0.02 kcal mol-1A-1). The epitope corresponding residues are color-coded (FIG. 11):Major Immunogenic Epitopes

1. SPOTs 86-92 aa 265PRKLNERLSLARANSV280-green2. SPOTs 102-105 aa 307ADNKTKEGRAMNR319-dark blue3. SPOTs 107-108 aa 319RRVFATITGSRTV331-yellow4. SPOTs 40-41 aa 121KYDFDGVNRGTRG133-violet (bottom right)

Example VI Comparison of the ORF Sequence, Epitope Sequences, andImmunogenicity of Previously Reported Sequences with the VaccineSequences Provided by the Invention

a. Alignment of the Prior Art Sequence with 6 Clinical IsolatesHarvested Between 1951 (ATCC17978) and 2009 (HUMC Strains)

The prior art sequence differs by 53/350 amino acids plus has anadditional 28 amino acids at the beginning of the sequence which doesnot appear in any A. baumannii OmpA sequence. In total, therefore, theprior art sequence differs by 81 amino acids (23% sequence divergence)vs. all 6 clinical isolates of A. baumannii used to infect mice.Finally, when compared to the sequences of 12 other A. baumanniiisolates in PubMed Genbank, the prior art sequence remains divergent(compare prior art sequence to the 12 aligned sequences in Table 4).

b. Sequences of Known B and T Cell Epitopes Vs. A. baumannii OmpASequences from ATCC 17978 and HUMC Strains Used to Infect Mice.

Comparing the amino acid sequences of the T and B cell epitopesidentified as immunodominant in the OmpA vaccine reveals that virtuallyevery immunodominant epitope has a different sequence than is present inthe prior art. Thus, the mutations that are distinct between previouslyknown sequences and the sequences of the present invention arespecifically present in the immune-reactive T and B cell epitopes (FIG.13).

c. Immunological Differences.

To determine if the sequence difference between previously knownsequences and the OmpA sequences of the invention result inimmunological differences, we infected 10 mice with sublethal inocula ofA. baumannii ATCC17978. Two weeks after infection, we harvested immunesera. ELISA plates were coated with OmpA that was either produced from asynthetic gene encoding a previously known sequence or produced from theOmpA sequence of the invention. The antibody titers of serum frominfected/immune mice were compared when the ELISA was run against theclaimed OmpA sequence versus the previously known sequence. Immune serumhad significantly higher titers against the OmpA than the patented OmpA(synthetic OmpA, or SOmpA, see FIG. 14). Median [IQ range] titers were12,800 [12,800-25,600] vs. 3,300 [1,600-8,000], p=0.002.

Example VII Monoclonal Antibodies (MAbs) Against OmpA Effectively TreatLethal A. baumannii Bloodstream Infection

Multiple MAbs were raised against OmpA and pre-clones selected forsubcloning by identifying those pre-clones that could bind to nativeOmpA on the A. baumannii surface. After selection by ELISA and flowcytometry for cell surface staining, five hybridoma subclones, 3 IgMsand 2 IgGs, were obtained. Hybridoma supernatants were dialyzed againstPBS. Negative control was an IgG isotype control MAb. C3H/FeJ mice wereinfected via the tail-vein with A. baumannii HUMC1 were treated IP with50 μg of MAb several hours after infection. 4 of the MAbs substantiallyimproved survival of infected mice, whereas 1 MAb was of no benefit (IgM#1) (FIG. 15). These data confirm that MAb therapy is effective againstthese infections, validating the concept of passive vaccination againstA. baumannii, and the composition of matter of the MAbs in hand.

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1-27. (canceled)
 28. A composition comprising an antibody, or antigenbinding fragment thereof, wherein said antibody or antigen bindingfragment specifically binds to an epitope encoded by an amino acidsequence selected from SEQ ID NOS:1-6.
 29. The composition of claim 28,wherein said epitope consists of at least 10 but less than 25 aminoacids in length and an amino acid sequence of SEQ ID NOS:1-6 thatcomprises: (i) the asparagine at position 91; (ii) the serine atposition 28; or (iii) the alanine at position
 163. 30. The compositionof claim 29, wherein said epitope consists of at least 10 but less than20 amino acids in length.
 31. The composition of claim 29, wherein saidepitope consists of at least 10 but less than 15 amino acids in length.32. The composition of claim 29, further comprising a pharmaceuticallyacceptable carrier.
 33. A method of prophylactic or therapeutictreatment of A. baumannii infection in a subject, comprisingadministering to the subject: (i) a composition comprising an antibody,or antigen binding fragment thereof, wherein said antibody or antigenbinding fragment specifically binds to an epitope encoded by an aminoacid sequence selected from SEQ ID NOS:1-6; or (ii) an immunologicallyeffective amount of a vaccine composition comprising an A. baumanniiouter membrane protein A (OmpA), or an antigenic fragment thereof,wherein said OmpA is at least 80% identical to SEQ ID NOS:1-6.
 34. Themethod of claim 33, wherein said epitope or said antigenic fragmentconsists of at least 10 but less than 25 amino acids in length and anamino acid sequence of SEQ ID NOS:1-6 that comprises: (i) the asparticacid at position 123, the aspartic acid at position 125, and theasparagine at position 128; (ii) the asparagine at position 91; (iii)the serine at position 28; or (iv) the alanine at position
 163. 35. Themethod of claim 34, wherein said epitope or said antigenic fragmentconsists of at least 10 but less than 20 amino acids in length.
 36. Themethod of claim 34, wherein said epitope or said antigenic fragmentconsists of at least 10 but less than 15 amino acids in length.
 37. Themethod of claim 33, wherein the subject is a human.
 38. An isolatedpolypeptide comprising an amino acid sequence selected from SEQ IDNOS:1-6 or an antigenic fragment thereof.
 39. The isolated polypeptideof claim 38, wherein said antigenic fragment consists of at least 10 butless than 25 amino acids in length and an amino acid sequence of SEQ IDNOS:1-6 that comprises: (i) the aspartic acid at position 123, theaspartic acid at position 125, and the asparagine at position 128; (ii)the asparagine at position 91; (iii) the serine at position 28; or (iv)the alanine at position
 163. 40. The isolated polypeptide of claim 39,wherein said antigenic fragment consists of at least 10 but less than 20amino acids in length.
 41. The isolated polypeptide of claim 39, whereinsaid antigenic fragment consists of at least 10 but less than 15 aminoacids in length.
 42. A vaccine composition comprising the isolatedpolypeptide of claim 38 and a pharmaceutically acceptable carrier. 43.The vaccine composition of claim 42, wherein said composition furthercomprises an adjuvant.
 44. An isolated nucleic acid molecule encodingthe isolated polypeptide, or the antigenic fragment thereof, of claim38.
 45. A vector comprising the isolated nucleic acid molecule of claim44.
 46. A vaccine composition comprising the vector of claim 45.